Prognostic value of preoperative serum alkaline phosphatase for predicting 3-year mortality in patients undergoing kidney transplantation: A retrospective study

Serum alkaline phosphatase (ALP) levels are related to high-turnover bone disease and reflect vascular calcification and inflammation. ALP has been reported to have a prognostic impact in various cohorts including chronic kidney disease. This study investigated whether preoperative serum ALP level could be used for predicting mortality in patients undergoing kidney transplantation. We retrospectively reviewed 1,718 patients who underwent kidney transplantation between November 2005 and June 2017. Finally, 1,533 patients who met the inclusion criteria were classified into tertiles based on preoperative serum ALP level (< 51, 51–72, > 72 IU/L). The incidence of mortality was compared among the three tertiles, and a stepwise logistic regression analysis was performed to evaluate the predictors for mortality. The incidence of 3-year mortality was the highest in the third tertile (1.0% vs. 2.5% vs. 4.4% in the first, second, and third tertile, respectively, p = 0.003). The third tertile of ALP level (odds ratio [OR] 1.855, 95% CI 1.192–2.886, p = 0.006), age (OR 1.052, 95% CI 1.022–1.082, p = 0.011), and history of hypertension (OR 0.401, 95% CI 0.210–0.765, p = 0.006) remained as independent predictors of mortality. Preoperative serum ALP level was significantly higher in the non-survivor group than in the survivor group (58.00 [44.00–76.00] vs. 75.00 [56.25–113.00], p = 0.003). The optimal cut-off value of serum ALP to predict 3-year mortality was 71 IU/L (area under the curve 0.636, 95% CI 0.554–0.719, p = 0.003). Therefore, preoperative serum ALP level was an independent predictor of 3-year mortality in patients undergoing kidney transplantation.


Introduction
The definite treatment for end-stage renal disease is kidney transplantation. Despite the development and recent advances in kidney transplantation, patients with chronic kidney disease (CKD) are highly prone to have a poor prognosis due to complications from CKD itself and various comorbidities. However, there are a few reports on risk stratifications for postoperative morbidity and mortality in patients undergoing kidney transplantation.
Serum alkaline phosphatase (ALP) is frequently used as a biomarker of high-turnover bone disease, and is known to be related to vascular calcification and inflammation [1,2]. In this context, ALP has been demonstrated to have a prognostic impact among older adults [3], and patients with coronary artery disease [4][5][6], and stroke [7]. Furthermore, ALP has been used to monitor bone metabolism and has a prognostic impact in patients with renal insufficiency whose mineral metabolism is altered [8]. Several studies have reported the association between ALP and mortality in patients with CKD and those on dialysis [9][10][11][12]. ALP predicts the prognosis of patients with CKD with altered mineralization and mineral metabolism changes after kidney transplantation. However, whether ALP may function as a predictor of prognosis in patients with CKD undergoing kidney transplantation has never been studied before.
Therefore, we aimed to conduct a retrospective study to investigate the association between preoperative serum ALP level and mortality in patients undergoing kidney transplantation.

Study population
This study was approved by our Institutional Review Board (IRB protocol No. 3-2021-0129), and the need for informed consent was waived due to the retrospective nature of the study. All data were fully anonymized before we accessed. We retrospectively reviewed the electronic medical records of patients who underwent kidney transplantation from November 2005 to June 2017 at the Severance Hospital. Out of 1,718 patients, 185 patients were excluded owing to data unavailability. A total of 1,533 patients were analyzed (Fig 1). Patients were divided into tertiles based on preoperative ALP levels (1 st

Study endpoints
The primary outcome of this study included the incidence of 3-year mortality after kidney transplantation. Secondary outcomes included the incidence of graft failure, new-onset diabetes after kidney transplantation (NODAT), myocardial infarction (MI), stroke, and acute kidney injury (AKI). Stroke was defined as the presence of newly developed neurological deterioration due to embolic, thrombotic, or hemorrhagic brain injury. AKI was defined as an increase in serum creatinine � 0.3 mg/dL, an increase to 150% of baseline value, or urine output <0.5 ml/kg/h for � 6 h consecutively within 48 h after kidney transplantation.

Statistical analyses
All statistical analyses were performed using the Statistical Package for Social Sciences version 23 (IBM Corp, Armonk, NY, USA). After normality assessment using the Kolmogorov-Smirnov test, the independent t-test or Mann-Whitney test was used to compare continuous variables. To compare the three groups, one-way ANOVA or Kruskal-Wallis test was used, as appropriate. Categorical variables were compared using the chi-squared or Fisher's exact test. Results are presented as means ± standard deviation (SD) for normally distributed data, medians (IQR) for skewed data, or number of patients (%). We performed logistic regression analysis to evaluate predictors of 3-year mortality after kidney transplantation with known risk factors and variables with p<0.2 depending on patient mortality. For multivariate analysis, a stepwise selection method was used and variables with p<0.2 in univariate analysis were selected. Predictability was expressed as odds ratio (OR) and 95% CI. Receiver-operating characteristic (ROC) curve analysis was used to determine the optimal cut-off value of preoperative serum ALP showing the best discriminatory capacity to predict postoperative 3-year mortality. The optimal cut-off value was defined as the serum ALP level showing the greatest sum of sensitivity and specificity. Multivariate logistic regression analysis was performed to find variables associated with ALP higher than the cut-off value we found (�71 IU/L). A p-value < 0.05 was considered statistically significant.

Results
A total of 1,718 patients were screened, and 1,533 patients were finally included for the analysis (Fig 1). Patients were classified into tertiles based on the preoperative ALP level ( The incidence of postoperative complications including the requirement of RRT (0.4% vs. 0.8% vs. 2.4%, p = 0.008) and AKI (0.6% vs. 1.1% vs. 2.4%, p = 0.043) was significantly higher in the third tertile than in the lower two tertiles. The incidence of 1-year (0.6% vs. 1.3% vs. 3.0%, p = 0.009) and 3-year mortality (1.0% vs. 2.5% vs. 4.4%, p = 0.003) was the highest in the third tertile group while the incidence of short-term mortality was similar among the groups ( Table 2).
In a multivariate logistic regression analysis to find possible associated factor with higher ALP, higher PTH was a predictor of higher ALP (�71 IU/L) ( Table 6).

Discussion
In this retrospective study, we demonstrated that preoperative serum ALP level could predict 3-year mortality of patients after kidney transplantation. The incidence of 1-year and 3-year mortality after kidney transplantation was the highest in the third tertile of ALP, whereas the incidence of 1-month and 6-month mortality among tertiles showed no significant differences. Moreover, the third tertile of ALP was demonstrated to be an independent predictor for developing 3-year mortality after kidney transplantation.
ALP is a hydrolase enzyme that dephosphorylates various molecules expressed in the bone, liver, placenta, and kidneys. Serum ALP is a biochemical marker of bone turnover or liver disease. There are four ALP isozymes (tissue-nonspecific isozyme, intestinal-type, placental-type, and placental-like). Among these isozymes, tissue-nonspecific ALP is the most abundant isoform and is involved in skeletal mineralization [13]. Since mineral metabolism is commonly altered in patients with CKD, ALP is frequently used to monitor bone metabolism associated with renal insufficiency [8].
Disorders related to calcium, phosphorous, and parathyroid hormone (PTH) are commonly observed in patients with CKD. Along with renal osteodystrophy, secondary hyperparathyroidism is common [14], and it may be associated with abnormal mineral metabolism leading to vascular calcification and poor prognosis [15][16][17]. Serum ALP levels have been demonstrated to be associated with mortality in patients with renal failure [11,12,18]. Serum ALP �120 IU/L was reported to be associated with mortality among patients undergoing hemodialysis [18]. The highest ALP quartile was associated with higher risk of mortality in patients undergoing peritoneal dialysis [12]. Although the association of abnormal mineral metabolism and CKD have been well established and the prognostic role of ALP in these patients has been demonstrated, the prognostic role of ALP in patients after kidney transplantation has not been investigated before. Further, bone turnover and abnormal bone mineralization showed changes even after kidney transplantation [19,20]. Thus, the strength of our study is that we investigated the prognostic role of ALP in patients undergoing kidney transplantation, rather than patients with CKD.
The mechanisms responsible for the association between elevated ALP levels and mortality after kidney transplantation are unclear. The first possible explanation is that ALP is a marker of high-turnover bone disease. Elevated serum PTH induces bone resorption and this can be manifested by elevated bone ALP levels [21]. Similarly, the PTH level showed significant differences between the groups according to the ALP tertile and higher PTH was an independent predictor of higher ALP (�71 IU/L) in the logistic regression analysis in the current study. A previous study demonstrated that mortality prediction by ALP was likely due to renal osteodystrophy [22]. Further, secondary hyperparathyroidism was associated with increased mortality in patients with CKD [23]. From these results, it can be inferred that ALP levels are increased due to renal osteodystrophy and secondary hyperparathyroidism in patients with CKD, and it can be associated with increased mortality. Furthermore, ALP levels have been demonstrated to be related with vascular calcification. A previous in vitro study revealed that vascular damage induces expression of tissue-nonspecific ALP and that ALP can promote calcification by hydrolyzing inorganic pyrophosphates [24]. An association between ALP and vascular calcification was also demonstrated in previous clinical studies [1,25]. Another possibility is the association of ALP and systemic inflammation. Elevated ALP levels were shown to be related to elevated C-reactive protein levels, indicative of systemic inflammation [26]. Further, inflammatory stimuli could lead to cellular responses, which could increase ALP expression [13]. Along these lines, a previous study reported an association between higher serum ALP level and infection-related mortality in patients undergoing peritoneal dialysis [27].
The optimal cut-off value we found through ROC curve analysis was 71 IU/L, which was similar to the cut-off value of the highest ALP tertile (>72 IU/L), which was considerably lower than the value obtained in previous studies investigating the association between ALP and mortality in patients undergoing dialysis. ALP �120 IU/L was reported to be associated with mortality in patients undergoing hemodialysis [18]. Another study conducted in patients undergoing peritoneal dialysis showed that the highest tertile of ALP (>155 IU/L) was associated with an infection-related mortality [27]. A previous study on the association between pretransplant ALP levels and mortality reported that ALP >120 IU/L was associated with an increased risk of mortality [28]. One notable difference is that the incidence of graft failure was significantly different according to the ALP level in the previous study, but not in our study.
As calcium-phosphate-PTH homeostasis was found to be a predictor of graft rejection in previous studies [29,30], there may be some correlation between elevated ALP, graft rejection, and increased mortality, which were not addressed in this study. This is because the cut-off value obtained in our study was not high enough to predict graft failure although it could predict mortality. In addition, since there is literature evidence demonstrating an increase in the ALP value itself rather than based on the specific cut-off value as a risk factor for mortality [12], the lower cut-off value obtained in the current study seems plausible.
Notably, ALP could predict long-term (1-year and 3-year) mortality rather than short-term mortality (1-month and 6-month) in this study. Considering that ALP levels are indicative of high-turnover bone disease, the cause of this prediction of long-term mortality may be a reflection of the severity of pre-existing disorders of bone and mineral metabolism. Because there are post-transplant bone disorders, which indicates pathologic processes occurring after transplantation that are superimposed on pre-existing disorders of bone and mineral metabolism secondary to kidney failure and/or diabetes mellitus [20]. Further, there have been several reports investigating postoperative changes in bone histomorphometry [19,31] supporting this hypothesis, which is beyond the scope of this study.
The strengths of our study are that we found the prognostic value of ALP in patients undergoing kidney transplantation rather than patients with CKD and this was not reported before. Further, we included a relatively long-term follow-up period and found that ALP levels might predict long-term mortality other than short-term mortality, and we identified a relatively lower cut-off value, which was within normal limits.
The current study has several limitations. First, possible confounding factors such as liver disease, inflammatory status, and comorbidities were not adjusted. However, since ALP levels are affected by various factors, it is worthwhile to divide patients based on the ALP tertile and compare the characteristics accordingly. Second, the inability to distinguish various isoforms of tissue non-specific ALP such as bone-specific ALP may be another limitation. However, clinical utility of measuring various isoforms of ALP remains unclear [32]. Further, many previous studies on ALP targeted total ALP rather than specific isoforms [3,4,7,12], and a previous study revealed that bone-specific ALP had weaker association with mortality than total ALP [9]. Lastly, there is a possibility that the recruited data are insufficient, resulting in a selection bias due to the retrospective nature of the study.

Conclusions
In conclusion, preoperative serum ALP level was an independent predictor of 3-year mortality after kidney transplantation. This retrospective study also revealed that ALP levels can predict long-term mortality rather than short-term mortality after kidney transplantation, which was not investigated before. Our results suggest that ALP level has a prognostic impact on mortality of patients after kidney transplantation, and that the possible mechanisms for this might be associated with high-turnover bone disease, vascular calcification, and inflammation.

Author Contributions
Conceptualization: Young Song, Sung Yeon Ham.